Abstract

BACKGROUND:

Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R).

RESULTS:

Overexpression of fusion proteins by IPTG induction caused significant differential expression of numerous cellular proteins; most of these proteins were down-regulated, including enzymes connected to the pentose phosphate pathway and the enzyme LuxS that could lead to an inhibition of tRNA synthesis. Interestingly, when plasmid-harboring cells were cultured in LB medium, gluconeogenesis occurred mainly through MaeB, while in the host strain, gluconeogenesis occurred by a different pathway (by Mdh and PckA). Significant up-regulation of the chaperones ClpB, HslU and GroEL and high-level expression of two protective small heat shock proteins (IbpA and IbpB) were found in cells overexpressing GST-GlcNAc 2-epimerase-5D but not in GST-Neu5Ac aldolase-5R-expressing E. coli. Although most of the recombinant protein was present in insoluble aggregates, the soluble fraction of GST-GlcNAc 2-epimerase-5D was higher than that of GST-Neu5Ac aldolase-5R. Also, in cells overexpressing recombinant GST-GlcNAc 2-epimerase-5D, the expression of σ32 was maintained at a higher level following induction.

CONCLUSIONS:

Differential expression of metabolically functional proteins, especially those in the gluconeogenesis pathway, was found between host and recombinant cells. Also, the expression patterns of chaperones/heat shock proteins differed among the plasmid-harboring bacteria in response to overproduction of recombinant proteins. In conclusion, the solubility of overexpressed recombinant proteins could be enhanced by maintaining the expression of σ32, a bacterial heat shock transcription factor, at higher levels during overproduction.

The glycolytic pathway, the gluconeogenic pathway, the TCA cycle, and the pentose phosphate pathway in E. coli cultured in LB medium. Up- and down-regulated proteins resulting from the proteome profiling are marked with ⊕ and ⊖, respectively.

Assay of expressed proteins by SDS-PAGE and ELISA. SDS-PAGE of protein extracts from E. coli BL21 harboring pGEX-2TK-nanA-5R after 3 h of IPTG induction (A), pGEX-2TK-2ep-5D (B) and pGEX-2TK (C). Quantification of GST-fused proteins in each extraction was done by ELISA using an anti-GST antibody (D). The molecular weights of GST-Neu5Ac aldolase-5R, GST-GlcNAc 2-epimerase-5D, and GST are 59, 70 and 28 kDa, respectively, and the positions are indicated by arrows. Lane M: protein marker; lanes 1-3: the first, second and third extractions from cell pellets; lane P: extraction from aggregates as described in the Methods section. Soluble percentage was defined as the percentage of total soluble protein in extractions 1, 2 and 3 as a function of the sum of total soluble protein and insoluble protein (extraction P). Error bars stand for S.D.

Expression of heat shock proteins in the host and recombinant E. coli. Expression levels of heat shock proteins prior to induction (upper panel) and at 3 h post-induction (lower panel) in the host strain and recombinant protein expressing strains. Abbreviations are as follows: BL, E. coli BL21; 2TK, E. coli BL21 harboring pGEX-2TK; NA, E. coli BL21 harboring pGEX-2TK-nanA-5R; EP, E. coli BL21 harboring pGEX-2TK-2ep-5D. The % volume was defined as the value of the intensity integration over the feature area of one spot divided by the total intensity integration over all of the spots in the whole gel image (100%). Error bars stand for S.D.

Western blot analysis of sigma 32 in recombinant protein expressing strains. Western analyses were performed for sigma 32 expressions in GST-, GST-GlcNAc 2-epimerase-5D-, and GST-Neu5Ac aldolase-5R-expressing E. coli BL 21 induced for 0, 1, 2, and 3 h. The samples marked with C (in upper panel) referre to the protein mixture loaded in each gel to serve as the inter-membrane control. In the graphical plot, the arbitrary unit was calculated by dividing the image intensity of the anti-σ32 band on developed film by that of the identical control on the same film. Error bars stand for S.D. (n = 3).